Mitochondrial Apoptotic Signaling Research Articles

Mitochondria and cell death.

Mitochondria are cellular factories for energy production, calcium homeostasis and iron metabolism, but they also have an unequivocal and central role in intrinsic apoptosis through the release of cytochrome c. While the subsequent activation of proteolytic caspases ensures that cell death proceeds in the absence of collateral inflammation, other phlogistic cell death pathways have been implicated in using, or engaging, mitochondria. Here we discuss the emerging complexities of intrinsic apoptosis controlled by the BCL-2 family of proteins. We highlight the emerging theory that non-lethal mitochondrial apoptotic signalling has diverse biological roles that impact cancer, innate immunity and ageing. Finally, we delineate the role of mitochondria in other forms of cell death, such as pyroptosis, ferroptosis and necroptosis, and discuss mitochondria as central hubs for the intersection and coordination of cell death signalling pathways, underscoring their potential for therapeutic manipulation.

Delivery of dental pulp stem cells by an injectable ROS-responsive hydrogel promotes temporomandibular joint cartilage repair via enhancing anti-apoptosis and regulating microenvironment

Temporomandibular joint (TMJ) cartilage repair poses a considerable clinical challenge, and tissue engineering has emerged as a promising solution. In this study, we developed an injectable reactive oxygen species (ROS)-responsive multifunctional hydrogel (RDGel) to encapsulate dental pulp stem cells (DPSCs/RDGel in short) for the targeted repair of condylar cartilage defect. The DPSCs/RDGel composite exhibited a synergistic effect in the elimination of TMJ OA (osteoarthritis) inflammation via the interaction between the hydrogel component and the DPSCs. We first demonstrated the applicability and biocompatibility of RDGel. RDGel encapsulation could enhance the anti-apoptotic ability of DPSCs by inhibiting P38/P53 mitochondrial apoptotic signal in vitro. We also proved that the utilization of DPSCs/RDGel composite effectively enhanced the expression of TMJOA cartilage matrix and promoted subchondral bone structure in vivo. Subsequently, we observed the synergistic improvement of DPSCs/RDGel composite on the oxidative stress microenvironment of TMJOA and its regulation and promotion of M2 polarization, thereby confirmed that M2 macrophages further promoted the condylar cartilage repair of DPSCs. This is the first time application of DPSCs/RDGel composite for the targeted repair of TMJOA condylar cartilage defects, presenting a novel and promising avenue for cell-based therapy.

Augmented mitochondrial apoptotic signaling impairs C2C12 myoblast differentiation following cellular aging through sequential passaging.

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self-renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age-related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high-passage (HP) myoblasts exhibit greater mitochondrial-mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase-9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts.

Shuangshen ningxin formula attenuates cardiac microvascular ischemia/reperfusion injury through improving mitochondrial function

Ethnopharmacological relevanceShuangshen Ningxin Formula (SSNX) is a traditional Chinese medicine formula used to treat myocardial ischemia–reperfusion injury (MIRI). A randomized controlled trial previously showed that SSNX reduced cardiovascular events, and experiments have also verified that SSNX attenuated ischemia–reperfusion (I/R) injury. However, the mechanism of SSNX in the treatment of microvascular I/R injury is still unclear. Aim of the studyTo determine whether SSNX protects the microvasculature by regulating I/R induction in rats and whether this effect depends on the regulation of NR4A1/Mff/Drp1 pathway. MethodsThe anterior descending coronary artery was ligated to establish a rat MIRI model with 45 min of ischemia and 24 h of reperfusion. The rats were subjected to a 7-day pretreatment with SSNX and nicorandil, after which their cardiac function and microvascular functional morphology were evaluated through diverse methods, including hematoxylin and eosin (HE) staining, wheat germ agglutinin (WGA) staining, and transmission electron microscopy. Cell apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Additionally, serum levels of ET-1 and eNOS were determined through an enzyme-linked immunosorbent assay (ELISA). The expression levels of NR4A1, Mff, and proteins related to mitochondrial fission were examined by Western blot (WB). Cardiac microcirculation endothelial cells (CMECs) were cultured and the oxygen–glucose deprivation/reoxygenation (OGD/R) model was duplicated. Following treatment with SSNX and DIM-C-pPhOH, an NR4A1 inhibitor, cell viability was assessed. Fluorescence was used to evaluate mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (MPTP) opening. Moreover, vascular endothelial function was evaluated through transendothelial electrical resistance (TEER), Transwell assays and tube formation assays. ResultsThe results showed that SSNX reduced the infarction area and no-flow area, improved cardiac function, mitigated pathological alterations, increased endothelial nitric oxide synthase expression, protected endothelial function, and attenuated microvascular damage after I/R injury. I/R triggered mitochondrial fission and apoptotic signaling in CMECs, while SSNX restored mitochondrial fission to normal levels and inhibited mitochondrial apoptosis. A study using CMECs revealed that SSNX protected endothelial function after OGD/R, attenuating the increase in NR4A1/Mff/Drp1 protein and inactivating VDAC1, HK2, cytochrome c (cyt-c) and caspase-9. Research also shows that SSNX can affect CMEC cell migration and angiogenesis, reduce mitochondrial membrane potential damage, and inhibit membrane opening. Moreover, DIM-C-pPhOH, an NR4A1 inhibitor, partially imitated the effect of SSNX. ConclusionSSNX has a protective effect on the cardiac microvasculature by inhibiting the NR4A1/Mff/Drp1 pathway both in vivo and in vitro.

Synthesis, crystal structure and antitumor activity of trialkyltin (IV) 9,10-anthraquinone-2-carboxylate

Two new anthraquinone-organotin complexes [Ph3Sn(O2CC14H7O2)·MeOH]·MeOH (AQS01) and Cy3Sn(O2CC14H7O2) (AQS02) were synthesized and characterized by elemental analysis, IR, 1H NMR, 13C NMR, 119 Sn NMR, X-ray crystallography and thermo-gravimetric analysis. Incorporation of 9,10-anthraquinone with organotin successfully enhanced its anticancer activity. Interestingly, the anti-proliferative activity of AQS01 against CAL-27 cells in vitro was almost 10-fold that of mitoxantrone and 40-fold that of cisplatin. Flow cytometry showed that AQS01 could induce apoptosis and arrest cell cycle in S phase. UV–visible absorption spectrometry and molecular docking showed that AQS01 can be well embedded in the double helix of DNA. Moreover, AQS01 induced cell death by upregulating the expression level of the DNA damage protein γ-H2AX and activating the mitochondrial apoptotic signaling pathway (Bax/Bcl-2). Therefore, AQS01 can be used as a potential anticancer drug and has potential for further research.

Tembusu virus induced apoptosis in vacuolate spermatogenic cells is mediated by Cytc-mediated mitochondrial apoptotic signaling pathway

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that infects mainly poultry and has caused huge economic losses to the poultry farming industry in China. Also known as duck hemorrhagic ovarian disease, DTMUV principally destroys ovarian tissue in ducks, causing a dramatic drop in egg production. and can also invade the male reproductive system causing lesions. Currently, little research has been done to reveal the underlying mechanisms of reproductive dysfunction in ducks caused by DTMUV infection. In this study, histopathological analysis and electron microscopy of testes of ducks infected with DTMUV showed that DTMUV caused testicular atrophy and cytoplasmic vacuolation in ducks. Terminal Deoxynucleotidyl Transferase-Mediated Nick-End Labeling (TUNEL) staining and real-time quantitative PCR(RT-qPCR) results further indicated that DTMUV induced spermatogenic cells apoptosis. After DTMUV infection, a large amount of cytochrome c(Cytc) was released from the mitochondrial matrix into the cytoplasm, activating downstream target proteins and causing apoptosis. To sum up, DTMUV induces spermatogenic cell apoptosis through the Cytc-induced mitochondrial apoptosis pathway, our study provides evidence for DTMUV infection-induced male reproductive disorders.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Targeting Mitochondrial Apoptotic Priming State to Personalize Therapy for Relapsed Acute Myeloid Leukemia

Despite of remarkable changes in therapeutic landscape of AML in past 5 years, relapsed AML patients still succumb to disease. In majority of patients, the common cause of death ultimately is the emergence multi-drug resistance at relapse. Using patient-derived xenograft (PDX) as our models, we investigated the basis of multi-drug resistance and methods for identifying drug sensitivity in relapsed AML. We hypothesized that there is differential activation of drug-induced mitochondrial apoptotic signaling between treatment-naïve and relapsed myeloblasts. To study mechanisms of drug resistance in AML, we developed PDX models (n=18 models) of in vivo acquired resistance to single agents (BCL-2/MCL-1 inhibitor, FLT3 inhibitor, SMAC mimetics and BRD-4 inhibitor) of distinct mechanisms, which caused board chemoresistance across all models. We tested whether multidrug resistance is associated with reduction in apoptotic signaling, using BH3 profiling, a method for measurement of mitochondrial sensitivity to apoptosis via exposure to synthetic BH3 domain peptides, on matched pre- and post-resistant PDXs. Using BH3 profiling, we found that a reduction in mitochondrial apoptotic priming consistently accompanied the acquisition of multidrug resistance irrespective of the mechanism of drug action and genetic status of PDX models. Next we created pharmacologic sensitivity landscape for 22 AML PDX models by exposing splenic myeloblasts isolated after confirmed engraftment (>70% circulating hCD45+ blasts) to drug a drug panel (n=40) for 14 hours. We first demonstrate that ex vivo drug-induced mitochondrial sensitivity singling measured via dynamic BH3 profiling (DBP) segregates PDXs according to prior treatment. By using unsupervised hierarchical clustering we found that AML PDXs developed from treatment naïve patients clustered together and showed greater apoptotic priming responses to a larger number of targeted agents than PDXs from R/R patients, which also formed discrete clusters. We replicated drug-response patterns from PDX models in primary and relapsed AML patients by analyzing ex vivo sensitivity responses to >500 drugs across relapse and diagnosis patients (Malani et al, 2021 and Tyner et al, 2018). We show that human AML patients at relapse also broadly lose sensitivity to agents in a similar pan-resistant manner as PDX models. Using unbiased whole transcriptomic analysis of naïve and resistant PDXs (n=78 samples), we found common pathways of resistance where positive enrichment was observed for cytokine-cytokine receptor interactions, xenobiotics biodegradation, metabolic pathways, Hippo signaling, and Ras signaling in resistant compared to treatment-naïve PDXs. Conversely, mechanisms relating to DNA repair and replication, such as base excision repair, mismatch repair, and homologous recombination, were negatively enriched. Moreover, pathways associated with specific drug resistance were also observed. One of the known multi-drug resistance mechanisms to chemotherapy drugs in AML is increased expression of efflux pumps from the superfamily of ATP binding cassette. We found enrichment in ABC transporter family pathway signatures in all 3 PDX models, DFAM-61786, DFAM-15354, and DFAM-61345, resistant to quizartinib, birinapant, and JQ-1 and validated higher drug-efflux activity in quizartinib and birinapant resistant model using calcein-AM efflux assay. We finally tested whether DBP could predict in vivo response of patient myeloblasts in PDX models. We tested in vivo sensitivity to 5 drugs of disparate mechanisms of action: birinapant and LCL-161 (SMAC mimetics), JQ-1 (BRD-4 inhibitor), venetoclax (BCL-2 antagonist), and quizartinib (FLT-3 inhibitor) in 4-9 different PDX models each. Using ex vivo DBP assay, we identified drugs that prime not only treatment naïve PDXs but also R/R PDXs. We identified persistent in vivo activity in a drug-specific manner, with BH3 mimetics and HDAC inhibitors showing overlapping activity across different drug-resistant models. Overall, we demonstrate that acquired resistance to targeted therapy in AML is accompanied by common mechanism of reduction in mitochondrial priming along with drug-specific resistance mechanisms. Further, we find that, even in the context of a multiply-resistant PDX model, DBP can still identify therapeutic vulnerabilities that can be efficaciously exploited in vivo.

Dietary Chito-oligosaccharide attenuates LPS-challenged intestinal inflammation via regulating mitochondrial apoptotic and MAPK signaling pathway

To investigate the regulatory effects of Chito-oligosaccharide (COS) on the anti-oxidative, anti-inflammatory, and MAPK signaling pathways. A total of 40 28-day-old weaned piglets were randomly allotted to 4 equal groups [including the control group, lipopolysaccharide (LPS) group, COS group, and COS*LPS group]. On the morning of d 14 and 21, piglets were injected with saline or LPS. At 2 h post-injection, whole blood samples were collected on d 14 and 21, and small intestine and liver samples were collected and analyzed on d 21. The results showed that COS inhibited the LPS-induced increase of malondialdehyde (MDA) concentration and hepatic TNF-α cytokines. COS significantly increased the serum total antioxidant capability (T-AOC) value on d 14, and total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) activities in both serum and liver on d 21. Furthermore, it increased hepatic catalase (CAT) activity. COS also increased the LPS-induced decrease in serum IgG concentrations. Immunohistochemical analysis results showed that COS significantly increased the jejunal and ileal Caspase 3, and ileal CD4+ values challenged by LPS. Dietary COS decreased the LPS-induced jejunal and ileal BAX and CCL2 mRNA levels, markedly decreased ileal COX2 and SOD1 mRNA levels, while increasing ileal iNOS. Furthermore, COS significantly increased the LPS-induced jejunal and ileal p-P38 and MyD88, as well as jejunal P38, while it effectively suppressed jejunal JNK1, and jejunal and ileal JNK2, p-JNK1, and p-JNK2 protein expressions. These results demonstrated that COS could be beneficial by attenuating LPS-challenged intestinal inflammation via regulating mitochondrial apoptotic and MAPK signaling pathways.

Taurine alleviates high-fat-high-glucose-induced pancreatic islet β-cell oxidative stress and apoptosis in rat

The effect of taurine (TAU) as a specific regulatory mediator on pancreatic function in obese rats induced by a high-fat-high-glucose (HFHG) diet was investigated. We fed male Sprague-Dawley rats under different conditions, namely the control, HFHG, TAU, and HFHG + TAU treatment groups for 4 months. Compared with the HFHG group, TAU supplementation significantly reduced malondialdehyde levels and increased superoxide dismutase, total antioxidant capacity, and glutathione levels in the rat pancreas. In addition, TAU significantly decreased the level of reactive oxygen species, and markedly increased the activity of heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP-1), and nuclear factor erythrocyte-2-related factor 2 (Nrf2) in the rat pancreas. Notably, HFHG diet could induce pancreatic injury in the rats through the Nrf2/HO-1 signaling pathway and activate the mitochondrial channel-mediated apoptotic signaling pathway. The addition of TAU significantly improved the pancreatic tissue injury induced by the HFHG diet in the rats and reduced the protein expression of Caspase-3, Cleaved-caspase-3, Caspase-9 and Bcl-2 associated protein X (BAX), and increased the protein expression of B-cell lymphoma-2 (Bcl-2). In conclusion, this experiment confirmed that TAU could alleviate the oxidative stress and apoptosis induced by the HFHG diet in rat pancreatic β-cells.

Multicolor-Encoded DNA Framework Enables Specific and Amplified In Situ Detection of the Mitochondrial Apoptotic Signaling Pathway.

Monitoring the molecular activation networks of cellular processes through fluorescence imaging to accurately elucidate the signaling pathways of mitochondrial apoptosis and the regulation of upstream and downstream molecules remains a current major challenge. In this work, a multicolor-encoded tetrahedral DNA framework (meTDF) carrying two pairs of catalytic hairpins is synthesized to monitor the intracellular upstream manganese superoxide dismutase (MnSOD) mRNA and the downstream cytochrome c (Cyt c) molecules for specific and sensitive detection of the mitochondrial apoptotic signaling pathway. These two types of molecules can trigger catalytic hairpin assembly (CHA) reactions with accelerated reaction kinetics for the hairpin pairs confined on meTDF to show highly amplified fluorescence for sensitive and simultaneous detection of MnSOD mRNA and Cyt c with detection limits of 3.7 pM and 0.23 nM in vitro, respectively. Moreover, the high stability and biocompatibility of the designed meTDF can facilitate efficient delivery of the probes into cells to monitor intracellular MnSOD mRNA and Cyt c for specific detection of the mitochondrial apoptosis pathway regulated by different drugs. With the successful demonstration of their robust capability, the meTDF nanoprobes can thus open new opportunities for detecting cell apoptotic mechanisms for studying the corresponding apoptotic signaling pathways and for screening potential therapeutic drugs.

Manganese triggers persistent activation of the integrated stress response by inhibition of SIRT1 on deacetylation of GADD34

Overexposure to manganese (Mn) is conducive to neurodegenerative diseases and neuronal injury. Persistent activation of the integrated stress response (ISR) has a substantial impact on the etiology of neurodegenerative disorders by interfering with intracellular homeostasis. Nevertheless, the precise mechanism through which ISR engages in Mn-related neurotoxicity remains unclear. Sirtuin 1 (SIRT1), a typical NAD+-dependent protein deacetylase, which is known to participate in Mn-induced neuronal damage. Therefore, the aim of our study was to clarify how SIRT1 regulates persistent ISR activation in mouse hippocampal neuronal cells (HT-22 cells) exposed to various concentrations of Mn. We discovered that persistent ISR activation was engaged in Mn-triggered mitochondrial and exogenous apoptotic signaling pathways, which was attributed to the excessive phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Growth arrest and DNA damage-inducible protein 34 (GADD34) is known to be responsible for down-regulating the phosphorylation of eIF2α. However, Mn promoted GADD34 protein expression and its acetylation level. We further investigated the effect of SIRT1 on the acetylation of GADD34 by overexpressing and silencing SIRT1. We discovered that SIRT1 activation significantly declined the acetylation level of GADD34, thus alleviating persistent ISR activation-mediated neuronal apoptosis in HT-22 cells-treated with Mn. In summary, these results suggested that Mn induced persistent activation of the ISR by inhibition of SIRT1 on deacetylation of GADD34.

TRPC6 promotes daunorubicin-induced mitochondrial fission and cell death in rat cardiomyocytes with the involvement of ERK1/2-DRP1 activation

Daunorubicin (DNR-) induced cardiotoxicity seriously restricts its clinical application. Transient receptor potential cation channel subfamily C member 6 (TRPC6) is involved in multiple cardiovascular physiological and pathophysiological processes. However, the role of TRPC6 anthracycline-induced cardiotoxicity (AIC) remains unclear. Mitochondrial fragmentation greatly promotes AIC. TRPC6-mediated ERK1/2 activation has been shown to favor mitochondrial fission in dentate granule cells. The aim of the present study was to elucidate the effects of TRPC6 on daunorubicin- induced cardiotoxicity and identify the mechanisms associated with mitochondrial dynamics. The sparkling results showed that TRPC6 was upregulated in models in vitro and in vivo. TRPC6 knockdown protected cardiomyocytes from DNR-induced cell apoptosis and death. DNR largely facilitated mitochondrial fission, boosted mitochondrial membrane potential collapse and damaged debilitated mitochondrial respiratory function in H9c2 cells，these effects were accompanied by TRPC6 upregulation. siTRPC6 effectively inhibited these mitochondrial adverse aspects showing a positive unexposed effect on mitochondrial morphology and function. Concomitantly, ERK1/2-DRP1 which is related to mitochondrial fission was significantly activated with amplified phosphorylated forms in DNR-treated H9c2 cells. siTRPC6 effectively suppressed ERK1/2-DPR1 over activation, hinting at a potential correlation between TRPC6 and ERK1/2-DRP1 by which mitochondrial dynamics are possibly modulated in AIC. TRPC6 knockdown also raised the Bcl-2/Bax ratio, which may help to block mitochondrial fragmentation-related functional impairment and apoptotic signaling. These findings suggested an essential role of TRPC6 in AIC by intensifying mitochondrial fission and cell death via ERK1/2-DPR1, which could be a potential therapeutic target for AIC.

Oxymatrine inhibits TGF‑β1‑mediated mitochondrial apoptotic signaling in alveolar epithelial cells via activation of PI3K/AKT signaling.

Although pulmonary fibrosis (PF) causes respiratory failure and death, effective therapies for PF have not been developed. Oxymatrine (OMT), an active ingredient in the Chinese herb Sophora flavescens, exerts antifibrotic effects; however, its effect on PF remains unclear. The present study aimed to determine whether OMT decreases transforming growth factor-β1 (TGF-β1)-induced PF in human lung cancer A549 cells by inhibiting apoptosis and targeting the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. To construct a PF cell model, A549 cells were stimulated with TGF-β1. The experimental groups were as follows: control (untreated cells grown in complete medium), TGF-β1 (cells treated with 5 ng/ml TGF-β1), OMT (cells treated with 5 ng/ml TGF-β1 and 0.25, 0.50, or 1.00 mg/ml OMT), and OMT + LY294002 (cells treated with 5 ng/ml TGF-β1, 1.0 mg/ml OMT. and 25 µmol/l LY294002). The effects of OMT on cell morphology (via electron microscopy), apoptosis (via Annexin V-PI staining), mitochondrial apoptosis signaling [using JC-1 method to analyze mitochondrial membrane potential (MMP)], and Bcl-2, as well as Bax expression (via western blotting and reverse transcription-quantitative polymerase chain reaction), were analyzed. OMT significantly protected cells against TGF-β1-induced PF by inhibiting apoptosis. The specific manifestations were cell injury, as evidenced by morphological changes and decreased MMP. Following OMT treatment, the expression of the pro-apoptotic protein Bax increased, whereas that of the anti-apoptotic protein Bcl-2 decreased. The PI3K/AKT-specific inhibitor LY294002 significantly inhibited the ameliorative effects of OMT on TGF-β1-induced apoptosis. Collectively, OMT attenuated TGF-β1-mediated mitochondrial apoptosis of alveolar epithelial cells by activating the PI3K/AKT signaling pathway. Therefore, OMT may be a promising drug for PF treatment.

Neuroprotective Effects of a Multi-Herbal Extract on Axonal and Synaptic Disruption in Vitro and Cognitive Impairment in Vivo.

Alzheimer's disease (AD) is a multifactorial disorder characterized by cognitive decline. Current available therapeutics for AD have limited clinical benefit. Therefore, preventive therapies for interrupting the development of AD are critically needed. Molecules targeting multifunction to interact with various pathlogical components have been considered to improve the therapeutic efficiency of AD. In particular, herbal medicines with multiplicity of actions produce cognitive benefits on AD. Bugu-M is a multi-herbal extract composed of Ganoderma lucidum (Antler form), Nelumbo nucifera Gaertn., Ziziphus jujuba Mill., and Dimocarpus longan, with the ability of its various components to confer resilience to cognitive deficits. To evaluate the potential of Bugu-M on amyloid-β (Aβ) toxicity and its in vitro mechanisms and on in vivo cognitive function. We illustrated the effect of Bugu-M on Aβ25-35-evoked toxicity as well as its possible mechanisms to diminish the pathogenesis of AD in rat cortical neurons. For cognitive function studies, 2-month-old female 3×Tg-AD mice were administered 400 mg/kg Bugu-M for 30 days. Behavioral tests were performed to assess the efficacy of Bugu-M on cognitive impairment. In primary cortical neuronal cultures, Bugu-M mitigated Aβ-evoked toxicity by reducing cytoskeletal aberrations and axonal disruption, restoring presynaptic and postsynaptic protein expression, suppressing mitochondrial damage and apoptotic signaling, and reserving neurogenic and neurotrophic factors. Importantly, 30-day administration of Bugu-M effectively prevented development of cognitive impairment in 3-month-old female 3×Tg-AD mice. Bugu-M might be beneficial in delaying the progression of AD, and thus warrants consideration for its preventive potential for AD.

Electroacupuncture alleviates ciliary muscle cell apoptosis in lens-induced myopic guinea pigs through inhibiting the mitochondrial signaling pathway.

To investigate the effect of electroacupuncture (EA) on the mitochondria-dependent apoptotic signaling pathway in the ciliary muscle of guinea pigs with negative lens-induced myopia (LIM). Guinea pigs were randomly divided into normal control (NC) group, LIM group, LIM+SHAM acupoint (LIM+SHAM) group, and LIM+EA group. Animals in the NC group received no intervention, while those in other three groups were covered with -6.0 diopter (D) lenses on right eyes. Meanwhile, animals in the LIM+EA group received EA at Hegu (LI4) combined with Taiyang (EX-HN5) acupoints, while those in the LIM+SHAM group were treated at sham points. After treatments for 1, 2, and 4wk, morphological changes in ciliary muscles were observed with hematoxylin and eosin (H&E) staining and nick end labeling (TUNEL), and the expression of the mitochondrial apoptotic signaling pathway-related molecules in ciliary muscles was measured by real-time quantitative polymerase chain reaction (qPCR) and Western blot. Additionally, the adenosine triphosphate (ATP) contents were also determined in ciliary muscles. Axial length increased significantly in the LIM and LIM+SHAM groups and decreased in the LIM+EA group. The ciliary muscle fibers were broken and destroyed in both LIM and LIM+SHAM groups, whereas those in the LIM+EA group improved significantly. TUNEL assay showed the number of apoptotic cells increased in the LIM and LIM+SHAM groups, whereas reduced in the LIM+EA group. ATP contents showed a significant decrease in the LIM and LIM+SHAM groups, whereas increased after EA treatment. Compared with the NC group, the dynamin-related protein 1 (DRP1), Caspase3, and apoptotic protease activator 1 (APAF1) levels were significantly increased in the LIM group and decreased in the LIM+EA group. The results provide evidence of EA inhibiting the development of myopia by regulating the mitochondrial apoptotic signaling pathway.

Peripheral blood mononuclear cell mitochondrial copy number and adenosine triphosphate inhibition test in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction. This study aims to develop biomarkers for assessing mitochondrial dysfunction in patients with NAFLD. Mitochondrion-associated transcriptome analysis was performed. Peripheral blood mononuclear cells obtained from patients with NAFLD (69) and healthy controls (19) were used to determine the mitochondrial DNA (mtDNA) copy number. A mitochondrial inhibition substrate test (ATP assay) was performed in HepG2 cells using the patient serum. Hepatic mRNA transcriptome analysis showed that the gene expression related to mitochondrial functions (mitochondrial fusion, apoptotic signal, and mitochondrial envelope) increased in patients with steatohepatitis, but not in those with NAFL. Gene set enrichment analysis revealed that the upregulated expression of genes is related to the pathways of the tricarboxylic (TCA) cycle and deoxyribonucleic acid (DNA) replication in patients with steatohepatitis, but not in healthy controls. The mtDNA copy number in the peripheral blood mononuclear cells was 1.28-fold lower in patients with NAFLD than that in healthy controls (P <.0001). The mitochondrial inhibition substrate test showed that the cellular adenosine triphosphate (ATP) concentration was 1.2-fold times less in NAFLD patients than that in healthy controls (P <.0001). The mtDNA copy number and mitochondrial ATP inhibition substrate test demonstrated negative correlations with the degree of hepatic steatosis, whereas the ATP concentration showed a positive correlation with the mtDNA copy number. The mitochondrial copy number of peripheral blood mononuclear cells and mitochondrial ATP inhibition substrate can be used as biomarkers for assessing the mitochondrial dysfunction in patients with NAFLD.

Potential Biochemical Pesticide-Synthesis of Neofuranocoumarin and Inhibition the Proliferation of Spodoptera frugiperda Cells through Activating the Mitochondrial Pathway.

Furanocoumarins, the secondary metabolites of plants, are considered to be natural insecticides and fungicides because they prevent the invasion of plant pathogenic microorganisms and the predation of herbivorous insects. In this study, novel 2-arylfuranocoumarin derivatives were designed to synthesize by condensation, esterification, bromination, and Wittig reaction. The results showed an excellent photosensitive activity of 2-thiophenylfuranocoumarin (I34). Cell Counting Kit-8 detected that I34 could inhibit the proliferation of Spodoptera frugiperda (Sf9) cells in a time- and concentration-dependent manner under ultraviolet A (UV-A) light for 3 min. The inverted microscope revealed that cells treated with I34 swelled, the membrane was ruptured, and apoptotic bodies appeared. The flow cytometry detected that I34 could induce apoptosis of Sf9 cells, increase the level of intracellular reactive oxygen species (ROS), decrease the mitochondrial membrane potential, and block cell cycle arrest in the G2/M phase. Transmission electron microscopy detected cell mitochondrial cristae damage, matrix degradation, and mitochondrial vacuolation. Further enzyme activity detection revealed that the enzyme activities of apoptosis-related proteins caspase-3 and caspase-9 increased significantly (p < 0.05). Finally, Western blotting analysis detected that the phosphorylation level of Akt and Bad and the expression of the apoptosis inhibitor protein Bcl-XL were inhibited, cleaved-PARP and P53 were increased, and cytochrome C was released from the mitochondria into the cytoplasm. Moreover, under UV-A irradiation, I34 promoted the increase in ROS in Sf9 cells, activated the mitochondrial apoptotic signal transduction pathway, and finally, inhibited cell proliferation. Thus, novel furanocoumarins exhibit a potential application prospect as a biochemical pesticide.

Stellettin B-Induced Oral Cancer Cell Death via Endoplasmic Reticulum Stress-Mitochondrial Apoptotic and Autophagic Signaling Pathway.

Oral squamous cell carcinoma (OSCC) affects tens of thousands of people worldwide. Despite advances in cancer treatment, the 5-year survival rate of patients with late-stage OSCC is low at 50–60%. Therefore, the development of anti-OSCC therapy is necessary. We evaluated the effects of marine-derived triterpene stellettin B in human OC2 and SCC4 cells. Stellettin B dose-dependently decreased the viability of both cell lines, with a significant reduction in OC2 cells at ≥0.1 µM at 24 and 48 h, and in SCC4 cells at ≥1 µM at 24 and 48 h. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells were significantly observed at 20 µM of stellettin B at 48 h, with the overexpression of cleaved caspase3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, mitochondrial respiratory functions were ablated by stellettin B. Autophagy-related LC3-II/LC3-I ratio and Beclin-1 proteins were increased, whereas p62 was decreased. At 20 µM at 48 h, the expression levels of the endoplasmic reticulum (ER) stress biomarkers calnexin and BiP/GRP78 were significantly increased and mitogen-activated protein kinase (MAPK) signaling pathways were activated. Further investigation using the autophagy inhibitor 3-methyladenine (3-MA) demonstrated that it alleviated stellettin B-induced cell death and autophagy. Overall, our findings show that stellettin B induces the ER stress, mitochondrial stress, apoptosis, and autophagy, causing cell death of OSCC cells.

Lupeol Treatment Attenuates Activation of Glial Cells and Oxidative-Stress-Mediated Neuropathology in Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) signifies a major cause of death and disability. TBI causes central nervous system (CNS) damage under a variety of mechanisms, including protein aggregation, mitochondrial dysfunction, oxidative stress, and neuroinflammation. Astrocytes and microglia, cells of the CNS, are considered the key players in initiating an inflammatory response after injury. Several evidence suggests that activation of astrocytes/microglia and ROS/LPO have the potential to cause more harmful effects in the pathological processes following traumatic brain injury (TBI). Previous studies have established that lupeol provides neuroprotection through modulation of inflammation, oxidative stress, and apoptosis in Aβ and LPS model and neurodegenerative disease. However, the effects of lupeol on apoptosis caused by inflammation and oxidative stress in TBI have not yet been investigated. Therefore, we explored the role of Lupeol on antiapoptosis, anti-inflammatory, and antioxidative stress and its potential mechanism following TBI. In these experiments, adult male mice were randomly divided into four groups: control, TBI, TBI+ Lupeol, and Sham group. Western blotting, immunofluorescence staining, and ROS/LPO assays were performed to investigate the role of lupeol against neuroinflammation, oxidative stress, and apoptosis. Lupeol treatment reversed TBI-induced behavioral and memory disturbances. Lupeol attenuated TBI-induced generation of reactive oxygen species/lipid per oxidation (ROS/LPO) and improved the antioxidant protein level, such as nuclear factor erythroid 2-related factor 2 (Nrf2) and heme-oxygenase 1 (HO-1) in the mouse brain. Similarly, our results indicated that lupeol treatment inhibited glial cell activation, p-NF-κB, and downstream signaling molecules, such as TNF-α, COX-2, and IL-1β, in the mouse cortex and hippocampus. Moreover, lupeol treatment also inhibited mitochondrial apoptotic signaling molecules, such as caspase-3, Bax, cytochrome-C, and reversed deregulated Bcl2 in TBI-treated mice. Overall, our study demonstrated that lupeol inhibits the activation of astrocytes/microglia and ROS/LPO that lead to oxidative stress, neuroinflammation, and apoptosis followed by TBI.